The roles of protein conformational dynamics and allostery in function are well-known. However, the roles that interdomain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between interdomain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the interdomain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using (1) an active site mutant (E219Q) of MMP1, (2) MMP9, another member of the MMP family that increases the activity of MMP1, and (3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent interdomain dynamics may enable allosteric control of the MMP1 function.

• MeSH
• Escherichia coli metabolismus   MeSH
• fibrinogen metabolismus   MeSH
• hemopexin metabolismus   MeSH
• katalytická doména fyziologie   MeSH
• katalýza MeSH
• kinetika MeSH
• matrixová metaloproteinasa 1 metabolismus   MeSH
• proteinové domény fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

• MeSH
• aminokyseliny metabolismus   MeSH
• Aspergillus oryzae enzymologie   metabolismus   MeSH
• endonukleasy specifické pro jednořetězcové nukleové kyseliny metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• katalytická doména fyziologie   MeSH
• katalýza MeSH
• kinetika MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• substrátová specifita MeSH
• vazebná místa fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH